This application claims the benefit of priority under 35 U.S.C. xc2xa7119 of foreign applications EP Application No. 00123728-8, filed Oct. 31, 2000, and EP Application No. 01106308-8, filed Mar. 15, 2001, the contents of which are hereby incorporated by reference in their entireties.
This invention relates to a method for the analysis of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample using a protease from a Bacillus strain. The invention further relates to a method for the analysis of a (at least one) target nucleic acid component of a mixture of non-proteinaceous components, which comprise nucleic acids, and proteinaceous components whereby the mixture is derived from a biological sample comprising the steps of incubating the mixture with a (at least one) protease from a Bacillus strain, optionally amplifying the (at least one) target nucleic acid component, and determining or detecting the (at least one) target nucleic acid component.
Many biological substances, especially nucleic acids, present special challenges in terms of isolating them from their natural environment. On the one hand, they are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances e.g. after lysis of cells. This makes them difficult to isolate or to measure, in particular in biospecific assays which allow the detection of specific analytes, e.g. nucleic acids, or specific analyte properties and play a major role in the field of diagnostics and bioanalytics in research and development. Examples for biospecific assays are hybridisation assays, immuno assays and receptor-ligand assays. Hybridisation assays use the specific base-pairing for the molecular detection of nucleic acid analytes e.g. RNA and DNA. Hence, oligonucleotide probes with a length of 18 to 20 nucleotides may enable the specific recognition of a selected complementary sequence e.g. in the human genome. Another assay which entails the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method allows the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of desoxynucleotide triphosphates in several cycles.
As described above, before the biological substances may be analysed in one of the above-mentioned assays or used for other processes, it has to be isolated or purified from biological samples containing complex mixtures of different components as e.g. proteinaceous and non-proteinaceous components. Often, for the first steps, processes are used which allow the enrichment of the component of interest, e.g. the non-proteinaceous material such as nucleic acids. Frequently, these are contained in a bacterial cell, a fungal cell, a viral particle, or the cell of a more complex organism, such as a human blood cell or a plant cell. The component of interest can also be called a xe2x80x9ctarget componentxe2x80x9d.
To release the contents of said cells or particles, they may be treated with enzymes or with chemicals to dissolve, degrade or denature the cellular walls of such organisms. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as lysate. A problem often encountered during the lysis is that other enzymes degrading the non-proteinaceous component of interest, e.g. desoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the component of interest during lysis. These degrading enzymes may also be present outside the cells or may have been spatially separated in different cellular compartiments before the lysis and come now into contact with the component of interest. Other components released during this process may be e.g. endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy.
There are a variety of means to tackle this problem mentioned-above. It is common to use chaotropic agents as e.g. guanidinium thiocyanate or anionic, cationic, zwitterionic or non-ionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases which rapidly degrade these enzymes or unwanted proteins. However, this may produce another problem as the said substances or enzymes can interfere with reagents or components in subsequent steps.
Enzymes which can be advantageously used in such lysis or sample preparation processes mentioned-above are enzymes which cleave the amide linkages in protein substrates and which are classified as proteases, or (interchangeably) peptidases (See Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3). Proteases which have been used in the prior art are e.g. alkaline proteases (W098/04730) or acid proteases (U.S. Pat. No. 5,386,024). The protease which is widely used in the prior art for sample preparation for the isolation of nucleic acids is proteinase K from Tritirachium album (see e.g. Sambrook et al., 1989) which is active around neutral pH and belongs to a family of proteases known to the person skilled in the art as subtilisins. A subtilisin is a serine protease produced by Gram-positive bacteria or fungi.
Bacteria of the Bacillus species secrete two extracellular species of protease, a neutral or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase, referred to as subtilisin. A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, in which there is an essential serine residue at the active site (White, Handler, and Smith, 1973 xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, N.Y., pp. 271-272). The serine proteases have molecular weights in the 25,000 to 30,000 Da (Dalton) range. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. The alternative term, alkaline protease, reflects the high pH optimum of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest, 1977, Bacteriological Rev. 41: 711-753).
A wide variety of subtilisins have been identified (see e.g. Kurihara et al., 1972, J. Biol. Chem. 247: 5629-563 1; Stahl and Ferrari, 1984, J. Bacteriol. 158: 411-418; Vasantha et al., 1984, J. Bacteriol. 159: 811-819, Jacobs et al., 1985, Nucl. Acids Res. 13: 8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366: 421-430; Svendsen et al., 1986, FEBS Lett 196: 228-232; Meloun et al., 1985, FEBS. Lett. 183: 195-200) including proteinase K from Tritirachium album (Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366: 584-492). Subtilisins are well characterized by their primary as well as by their tertiary structure (see e.g. Kraut, 1977, Ann. Rev. Biochem. 46: 331-358; Kurihara et al., 1972, J. Biol. Chem. 247: 5629-5631; Stahl and Ferrari, 1984, J. Bacteriol. 158: 411-418; Vasantha et al., 1984, J. Bacteriol. 159: 811-819; Jacobs et al., 1985, Nucl. Acids Res. 13: 8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366: 421-430; Svendsen et al., 1986, FEBS Lett. 196: 228-232; Meloun et al., 1985, FEBS Lett. 183: 195-200; Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366: 485-492).
In connection with this invention the amino acid and DNA sequences of two further serine proteases are of particular interest. These proteases were derived from two Bacillus lentus variants, 147 and 309, which have been deposited with NCIB and designated the accession Nos. NCIB 10147 and NCIB 10309, respectively (see WO89/06279 and U.S. Pat. No. 3,723,250). For convenience the proteases produced by these strains are designated subtilisin 147 and subtilisin 309, respectively, and the genes encoding these proteins are referred to as the subtilisin 147 and 309 genes. The disclosure of these sequences can be found in WO89/06279. The equivalents thereto are EP396608 and U.S. Pat. No. 5,741,694. Subtilisins have found much utility in industry, particularly detergent formulations used for the washing of clothes.
In the next steps of the sample preparation which follow on the lysis step, the component of interest is further enriched. If the non-proteinaceous components of interest are e.g. nucleic acids, they are normally extracted from the complex lysis mixtures before they are used in a probe-based assay.
There are several methods for the extraction of nucleic acids:
sequence-dependent or biospecific methods as e.g.:
affinity chromatography
hybridisation to immobilised probes
sequence-independent or physico-chemical methods as e.g.:
liquid-liquid extraction with e.g. phenol-chloroform
precipitation with e.g. pure ethanol
extraction with filter paper
extraction with micelle-forming agents as cetyl-trimethyl-ammonium-bromide
binding to immobilised, intercalating dyes, e.g. acridine derivatives
adsorption to silica gel or diatomic earths
adsorption to magnetic glass particles (MGP) or organo silane particles under chaotropic conditions
Particularly interesting for extraction purposes is the adsorption of nucleic acids to a glass surface although other surfaces are possible. Many procedures for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to glass surfaces.
As mentioned above, the protease which is widely used in the prior art for sample preparation for the isolation of nucleic acids is proteinase K from Tritirachium album. However, this protease has the disadvantage that the production is relatively expensive. Further, proteinase K is disadvantageous in methods using magnetic glass particles for the nucleic acid isolation from EDTA, heparin or citrate blood plasma, as the particles will often stick to one another. This is very disadvantageous for automated processes used for the analysis of a very large number of samples.
Therefore, it was an object of the present invention to provide a new method for the analysis of target non-proteinaceous components, in particular nucleic acids, using a protease which is relatively cheap, has constant quality and can be used in a variety of processes. Preferably it should be possible to use it for the analyis of a (at least one) target nucleic acid component from a variety of different matrices e.g. EDTA, citrate, or heparin blood plasma or blood serum. This method should be particularly suitable in automated processes. Ideally the protease would be also very active in the presence of chaotropic agents frequently used in the processes for the purification of nucleic acids.
This problem was solved by the findings of the present invention which is related to a method for the analysis of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample comprising the step of incubating the mixture with a (at least one) protease having an amino acid sequence which is at least 80% identical to the amino acid sequence of the protease subtilisin 147 from Bacillus lentus. As can be seen from the example, the protease is very active in the presence of chaotropic agents or equally active for the digestion of citrate or EDTA blood plasma. This could not be foreseen from the prior art.
In summary, this invention relates to a method for the analysis of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample using a protease from a Bacillus strain. The invention further relates to a method for the analysis of a (at least one) target nucleic acid component of a mixture of non-proteinaceous components, which comprise nucleic acids, and proteinaceous components whereby the mixture is derived from a biological sample comprising the steps of incubating the mixture with a (at least one) protease having an amino acid sequence which is at least 80% identical to the amino acid sequence of the protease subtilisin 147 from Bacillus lentus, optionally amplifying the (at least one) target nucleic acid component, and determining or detecting the (at least one) target nucleic acid component. Optionally, the nucleic acids and the (at least one) target nucleic acid component are bound to a material with an affinity thereto, optionally washed and optionally released from the material with an affinity thereto, whereby the material with an affinity to nucleic acids and the (at least one) target nucleic acid component comprises a material with a silica surface, in particular magnetic glass particles. The invention is further related to the use of a protease according to the invention in diagnostics, research and bioanalytics e.g. for the purification of nucleic acids, for the analysis of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample, for the enrichment of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample or for the purification or isolation of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample. The invention is also related to a kit comprising the protease according to the invention and the use of a kit according to the invention in diagnostics and/or for the purification of nucleic acids. The invention will be described in more detail below.